[Show abstract][Hide abstract] ABSTRACT: The hop plant, Humulus lupulus L., has an exceptionally high content of secondary metabolites, the hop α-acids, which possess a range of beneficial properties,
including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic
acid bacteria. The present study investigated molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found
with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acid detoxification
and tolerance. This screening and further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves
three major processes, active proton pumping into the vacuole by the vacuolar-type ATPase to enable vacuolar sequestration
of iso-α-acids and alteration of cell wall structure and, to a lesser extent, active export of iso-α-acids across the plasma
membrane. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelators.
Full-text · Article · Nov 2009 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Accumulation of glycogen and trehalose in nutrient-limited cultures of Saccharomyces cerevisiae is negatively correlated with the specific growth rate. Additionally, glucose-excess conditions (i.e., growth limitation by nutrients other than glucose) are often implicated in high-level accumulation of these storage carbohydrates. The present study investigates how the identity of the growth-limiting nutrient affects accumulation of storage carbohydrates in cultures grown at a fixed specific growth rate. In anaerobic chemostat cultures (dilution rate, 0.10 h(-1)) of S. cerevisiae, the identity of the growth-limiting nutrient (glucose, ammonia, sulfate, phosphate, or zinc) strongly affected storage carbohydrate accumulation. The glycogen contents of the biomass from glucose- and ammonia-limited cultures were 10- to 14-fold higher than those of the biomass from cultures grown under the other three glucose-excess regimens. Trehalose levels were specifically higher under nitrogen-limited conditions. These results demonstrate that storage carbohydrate accumulation in nutrient-limited cultures of S. cerevisiae is not a generic response to excess glucose but instead is strongly dependent on the identity of the growth-limiting nutrient. While transcriptome analysis of wild-type and msn2Delta msn4Delta strains confirmed that transcriptional upregulation of glycogen and trehalose biosynthesis genes is mediated by Msn2p/Msn4p, transcriptional regulation could not quantitatively account for the drastic changes in storage carbohydrate accumulation. The results of assays of glycogen synthase and glycogen phosphorylase activities supported involvement of posttranscriptional regulation. Consistent with the high glycogen levels in ammonia-limited cultures, the ratio of glycogen synthase to glycogen phosphorylase in these cultures was up to eightfold higher than the ratio in the other glucose-excess cultures.
Full-text · Article · Oct 2009 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Saccharomyces cerevisiae has been used for at least eight millennia in the production of alcoholic beverages (41). Along with ethanol and carbon dioxide, fermenting cultures of this yeast produce many low-molecular-weight flavor compounds. These alcohols, aldehydes, organic acids, esters, organic sul- fides, and carbonyl compounds have a strong impact on prod- uct quality. Indeed, the subtle aroma balance of these com- pounds in fermented foods and beverages is often used as an organoleptic fingerprint for specific products and brands (42). Food fermentation by yeast and lactic acid bacteria is accom- panied by the formation of the aliphatic and aromatic alcohols known as fusel alcohols. Fusel oil, which derives its name from the German word fusel (bad liquor), is obtained during the distillation of spirits and is enriched with these higher alcohols. While fusel alcohols at high concentrations impart off-flavors, low concentrations of these compounds and their esters make an essential contribution to the flavors and aromas of fer- mented foods and beverages. Fusel alcohols are derived from amino acid catabolism via a pathway that was first proposed a century ago by Ehrlich (13). Amino acids represent the major source of the assimilable nitrogen in wort and grape must, and these amino acids are taken up by yeast in a sequential manner (23, 32). Amino acids that are assimilated by the Ehrlich path- way (valine, leucine, isoleucine, methionine, and phenylala- nine) are taken up slowly throughout the fermentation time (32). After the initial transamination reaction (Fig. 1), the resulting -keto acid cannot be redirected into central carbon metabolism. Before -keto acids are excreted into the growth medium, yeast cells convert them into fusel alcohols or acids via the Ehrlich pathway. Current scientific interest in the Ehrlich pathway is sup- ported by increased demands for natural flavor compounds such as isoamyl alcohol and 2-phenylethanol, which can be produced from amino acids in yeast-based bioconversion pro- cesses (14), as well as by the need to control flavor profiles of fermented food products. The goal of this paper is to present a concise centenary overview of the biochemistry, molecular biology, and physiology of this important pathway in S. cerevi- siae.
Full-text · Article · May 2008 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Transcriptional responses of the yeast Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under limiting and abundant Zn concentrations in chemostat
culture. To investigate the context dependency of this transcriptional response and eliminate growth rate-dependent variations
in transcription, yeast was grown under several chemostat regimens, resulting in various carbon (glucose), nitrogen (ammonium),
zinc, and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified, and the set
enabled the definition of the Zn-specific Zap1p regulon, comprised of 26 genes and characterized by a broader zinc-responsive
element consensus (MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent regulation of genes involved
in storage carbohydrate metabolism. Their concerted down-regulation was physiologically relevant as revealed by a substantial
decrease in glycogen and trehalose cellular content under Zn limitation. An unexpectedly large number of genes were synergistically
or antagonistically regulated by oxygen and Zn availability. This combinatorial regulation suggested a more prominent involvement
of Zn in mitochondrial biogenesis and function than hitherto identified.
Full-text · Article · Jan 2008 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Saccharomyces cerevisiae can use a broad range of compounds as sole nitrogen source. Many amino acids, such as leucine, tyrosine, phenylalanine and methionine, are utilized through the Ehrlich pathway. The fusel acids and alcohols produced from this pathway, along with their derived esters, are important contributors to beer and wine flavor. It is unknown how these compounds are exported from the cell. Analysis of nitrogen-source-dependent transcript profiles via microarray analysis of glucose-limited, aerobic chemostat cultures revealed a common upregulation of PDR12 in cultures grown with leucine, methionine or phenylalanine as sole nitrogen source. PDR12 encodes an ABC transporter involved in weak-organic-acid resistance, which has hitherto been studied in the context of resistance to exogenous organic acids. The hypothesis that PDR12 is involved in export of natural products of amino acid catabolism was evaluated by analyzing the phenotype of null mutants in PDR12 or in WAR1, its positive transcriptional regulator. The hypersensitivity of the pdr12Delta and war1Delta strains for some of these compounds indicates that Pdr12p is involved in export of the fusel acids, but not the fusel alcohols derived from leucine, isoleucine, valine, phenylalanine and tryptophan.
Full-text · Article · Oct 2006 · FEMS Yeast Research